TNT kit with T7 RNA Pol

Mark Guiltinan mark_guiltinan at AGCS.CAS.PSU.EDU
Mon Apr 1 18:06:53 EST 1996


We have found that the concentration of PCR template is very important with
the Promega TnT kit, but not so with other kits (Ambion).  You need to titrate
the template, too much will kill the reaction.  Here is an excerpt from a
paper we are about to publish (a short note).

PCR Reactions  PCR reactions were performed to amplify a region of the EmBP
gene spanning amino acid residues 150-308 as described (Guiltinan and Miller,
1994).  The 5' primer was designed to incorporate a T7 promoter and an
in-frame start codon into the final product.  The 3' primer has an in frame
stop codon and cloning sites 3' of the coding region.  PCR reactions were
performed in duplicate in a volume of 100 microliters containing 3 ng plasmid
template, 20 pmoles of each primer, 240 mM dNTPs, 2-5 U Taq polymerase
(Promega, Madison, WI), 2 mM MgCl2, and the appropriate 10X buffer supplied
with the enzyme.  Samples were subjected to a 5 minute "hot start" at 95#161#C
during which the dNTPs were added, followed by 35 cycles of denaturing at
95#161#C for 1 minute, annealing at 58#161#C for 1 minute, and extension at
72#161#C for 1.5 minutes; and a final 5 minute extension at 72#161#C.  At this
point, the duplicate samples were pooled and the 518 bp product purified using
Wizard PCR Preps (Promega, Madison, WI) and concentrated into a final volume
of 50 ul in TE (10 mM Tris, 1 mM EDTA, pH 7.6) buffer.  DNA concentrations
were assessed using a Gene Quant DNA Calculator (Pharmacia Biotech, Cambridge,
England).  
Transcription-Translation System   A T7 coupled Reticulocyte Lysate System
(TnT, Promega, Madison, WI) was utilized to perform the
transcription/translation using the PCR product templates according to the
manufacturers' instructions for 25 ul reactions.  Eight microliters of
undiluted PCR product, which contained 16 pmoles of DNA, were used in reaction
number one.  Reactions 2-7 contained 8 ul of sequential two-fold dilutions of
the PCR reaction product.  RNAse free water alone was used in reaction eight
to serve as the unprogrammed control.


	In order to test the effect of template concentration of the efficiency of
the TnT reaction system, a two fold dilution series of the PCR template was
prepared and equal volumes of each were added to TnT reactions.  Reaction
products were run on SDS gels to evaluate the amount of protein synthesis in
each reaction (Fig. 1).  As expected, the amount of protein synthesized was
proportional to the amount of template added at the lower concentrations,
raising to a maximum of about 1.8 pmol of template (corresponding to about 0.6
ug of DNA).  However, when larger amounts of template were added, protein
synthesis was inhibited.  In the highest concentration tested (7.5 pmol or
about 2.5 ug), protein was barely detectable.  Quantitation of the protein
product showed that the effect is quantitatively dramatic, resulting in a 4
fold reduction in protein product when 7.5 pmol is added (Fig. 1b).

	Following the manufacturers' instructions, if an investigator adds up to 2
ug/50 ul reaction (equivalent to 1 ug/25 ul reaction used in this study), of a
500 bp PCR fragment, this represents a final concentration of 3 pmol/25ul, 10
times higher than if a plasmid of 5 kbp were used and this can result in an
approximate 50% reduction in protein synthesized.  If one doubles this
concentration in a reaction, a nearly complete inhibition of protein synthesis
results.  It should also be noted that if one simply calculates the molarity
of the template based on the manufacturers instructions and assuming a 5 kbp
plasmid, only up to 0.3 pmol/25 ul reaction would be used, resulting in a
sub-optimal protein production.
	It is clear from these results that when considering using PCR templates for
TnT reactions, a titration of template over a range between 0.2 to 7.5 pmol/25
ul reaction should first be performed to optimize the production of protein
for each template.  With this consideration, PCR based-coupled
transcription-translation system is a powerful tool in the study of gene and
protein function in vitro.


Mark Guiltinan
Department of Horticulture
The Biotechnology Institute
Penn State University
306 Wartik Lab
University Park, PA 16802-5807
office:  814 863-7957
lab:       814 863-7958
fax:       814 863-6139
mjg at psupen.psu.edu



More information about the Methods mailing list